F. Del Galdo

A role for caveolin-1 in desmoglein binding and desmosome dynamics

Desmoglein-2 (Dsg2) is a desmosomal cadherin that is aberrantly expressed in human skin carcinomas. In addition to its well-known role in mediating intercellular desmosomal adhesion, Dsg2 regulates mitogenic signaling that may promote cancer development and progression. However, the mechanisms by which Dsg2 activates these signaling pathways and the relative contribution of its signaling and adhesion functions in tumor progression are poorly understood. In this study we show that Dsg2 associates with caveolin-1 (Cav-1), the major protein of specialized membrane microdomains called caveolae, which functions in both membrane protein turnover and intracellular signaling. Sequence analysis revealed that Dsg2 contains a putative Cav-1-binding motif. A permeable competing peptide resembling the Cav-1 scaffolding domain bound to Dsg2, disrupted normal Dsg2 staining and interfered with the integrity of epithelial sheets in vitro. Additionally, we observed that Dsg2 is proteolytically processed; resulting in a 95-kDa ectodomain shed product and a 65-kDa membrane-spanning fragment, the latter of which localizes to lipid rafts along with full-length Dsg2. Disruption of lipid rafts shifted Dsg2 to the non-raft fractions, leading to the accumulation of these proteins. Interestingly, Dsg2 proteolytic products are elevated in vivo in skin tumors from transgenic mice overexpressing Dsg2. Collectively, these data are consistent with the possibility that accumulation of truncated Dsg2 protein interferes with desmosome assembly and/or maintenance to disrupt cell–cell adhesion. Furthermore, the association of Dsg2 with Cav-1 may provide a mechanism for regulating mitogenic signaling and modulating the cell-surface presentation of an important adhesion molecule, both of which could contribute to malignant transformation and tumor progression.

The enhanced liver fibrosis test: a clinical grade, validated serum test, biomarker of overall fibrosis in systemic sclerosis

Objectives The absence of a serological surrogate outcome measure of fibrosis in systemic sclerosis (SSc) is a major deficiency for intervention studies and clinical management. An algorithm including the serum concentration of procollagen-III aminoterminal-propeptide, tissue inhibitor of matrix metalloproteinase-1 and hyaluronic acid, has recently been validated as predictive of severity and clinical outcome in chronic liver diseases (enhanced liver fibrosis (ELF) test) and implemented as a clinical grade test available for physicians. We evaluated the ELF test as a surrogate outcome measure in SSc. Methods The ELF score was determined blindly in 210 patients with SSc. Results were correlated with clinical, functional and instrumental variables including disease severity, activity and disability. Results The ELF test was above normal range in 83% of SSc patients (175/210). The ELF score showed a significant correlation (p<0.0001) with extent of skin involvement (r=0.28), diffusing lung capacity of carbon monoxide (DLCO) (r=−0.32), health assessment questionnaire–disability index (HAQ-DI) (r=0.32), disease severity score (r=0.3) and age (r=0.41). In addition, ELF correlated with disease activity (r=0.23; p=0.02). Using regression analysis, the extent of skin involvement, age, DLCO and gender were independently associated with the ELF score. The ELF score did not correlate with the presence of pulmonary artery hypertension, digital ulcers or any other measure of vasculopathy. Conclusions The ELF test is a clinical-grade serum test that significantly correlates with several measures of fibrosis in SSc and with overall disease activity, severity and HAQ-DI. The specific correlation with fibrosis and its face validity, together with the feasibility of the test, warrant its further development as a surrogate outcome measure of fibrosis in SSc.

Junctional adhesion molecule-A is abnormally expressed in diffuse cutaneous systemic sclerosis skin and mediates myeloid cell adhesion

Objective: To investigate the role of junctional adhesion molecule-A (JAM-A) in the pathogenesis of systemic sclerosis (SSc). Methods: Biopsy specimens from proximal and distal arm skin and serum were obtained from patients with SSc and normal volunteers. To determine the expression of JAM-A on SSc dermal fibroblasts and in SSc skin, cell surface ELISAs and immunohistology were performed. An ELISA was designed to determine the amount of soluble JAM-A (sJAM-A) in serum. Myeloid U937 cell–SSc dermal fibroblast and skin adhesion assays were performed to determine the role of JAM-A in myeloid cell adhesion. Results: The stratum granulosum and dermal endothelial cells (ECs) from distal arm SSc skin exhibited significantly decreased expression of JAM-A in comparison with normal volunteers. However, sJAM-A was increased in the serum of patients with SSc compared with normal volunteers. Conversely, JAM-A was increased on the surface of SSc compared with normal dermal fibroblasts. JAM-A accounted for a significant portion of U937 binding to SSc dermal fibroblasts. In addition, JAM-A contributed to U937 adhesion to both distal and proximal SSc skin. Conclusions: JAM-A expression is dysregulated in SSc skin. Decreased expression of JAM-A on SSc ECs may result in a reduced response to proangiogenic basic fibroblast growth factor. Increased JAM-A expression on SSc fibroblasts may serve to retain myeloid cells, which in turn secrete angiogenic factors.

There is a need for new systemic sclerosis subset criteria. A content analytic approach

Objectives. Systemic sclerosis (SSc) is heterogenous. The objectives of this study were to evaluate the purpose, strengths and limitations of existing SSc subset criteria, and identify ideas among experts about subsets. Methods. We conducted semi-structured interviews with randomly sampled international SSc experts. The interview transcripts underwent an iterative process with text deconstructed to single thought units until a saturated conceptual framework with coding was achieved and respondent occurrence tabulated. Serial cross-referential analyses of clusters were developed. Results. Thirty experts from 13 countries were included; 67% were male, 63% were from Europe and 37% from North America; median experience of 22.5 years, with a median of 55 new SSc patients annually. Three thematic clusters regarding subsetting were identified: research and communication; management; and prognosis (prediction of internal organ involvement, survival). The strength of the limited/diffuse system was its ease of use, however 10% stated this system had marginal value. Shortcomings of the diffuse/limited classification were the risk of misclassification, predictions/generalizations did not always hold true, and that the elbow or knee threshold was arbitrary. Eighty-seven percent use more than 2 subsets including: SSc sine scleroderma, overlap conditions, antibody-determined subsets, speed of progression, and age of onset (juvenile, elderly). Conclusions. We have synthesized an international view of the construct of SSc subsets in the modern era. We found a number of factors underlying the construct of SSc subsets. Considerations for the next phase include rate of change and hierarchal clustering (e.g. limited/diffuse, then by antibodies).

OP0228 Optical coherence tomography validation: A new quantitative imaging biomarker for affected skin in scleroderma

Background Skin involvement in systemic sclerosis (SSc) is often primary outcome in clinical trials but it is still orphan of a quantitative imaging technique. Optical Coherence Tomography (OCT) is an emerging imaging technology for clinical examination employing a low-intensity infra-red laser beam and providing high-contrast 2 mm deep skin images with a 4 micron resolution. Objectives The aim of this study was to evaluate face validity and reliability of OCT in SSc. Methods Dorsal aspect of forearms was assessed employing topical probe “VivoSight” (Michelson Diagnostics) and optics of Swept-source Fourier-Domain type with a laser wavelength of 1305±15 nm. Clinical skin involvement was determined using the mRSS. The study included 8 foreams from 8 healthy controls (HC) and 5 forearms for each group scored from “0” to “3” from 13 SSc patients. Hematoxylin-Eosin (H&E) staining was performed from forearm skin biopsy, within 1 cm of OCT scanned region, in one HC and one SSc patient (mRSS=3 on the site of analysis). Matlab software was employed to calculate mean optical density (OD) of the scans. Signal changes within epidermis (ED), Dermal-Epidermal Junction (DEJ) and dermis of SSc patients and HC were analysed and collated to a unique graph. To investigate the intraobserver and interobserver reproducibility, intraclass correlation coefficient (ICC) and limits of agreement (LoA) were calculated for the minimum value after the first peak and the maximum value of the second peak of the averaged A-scans. Results OCT images collected in HC showed a regular hyper-reflective border of the skin surface and a homogeneous hypo-reflective epidermal layer. The papillary dermis (PD) was visualized as hyper-reflective area compared to the adjacent ED allowing the visualization of the DEJ. Mean OD data showed that DEJ was a OD nadir region between 60 and 70 micron from the surface, the PD a high density region (OD range:0.64-0.72; micron range:60-100) and the reticular dermis (RD) had a OD ranging from 0.72 and 0.4. In contrast SSc affected tissues (mRSS=3) showed no DEJ and no increase in OD in the PD which appeared with a range density of 0.61-0.56. Interestingly in SSc non-affected tissues (mRSS=0), the valley corresponding to DEJ was still visible with an almost normal OD range of the PD (OD=0.64-0.67) and an OD of the second peak lower than healthy subjects. In the remaining cases (mRSS=1 and 2) the valley after the entrance peak was still visible, however with a weaker second peak compared to healthy skin and SSc non-affected skin. Validation with H&E staining confirmed the localisation of the above mentioned density areas.In addition the results showed excellent intraobserver reliability (ICC >0.98; LoA=0.94-0.99) in both parameters measured by the same clinician on the two separate occasions. The interobserver reliability was also excellent with ICC>0.89 (LoA=0.61-0.97) in both parameters assessed by two investigators. Conclusions This is a proof of concept validation of face validity of OCT as quantitative imaging technique of scleroderma skin. The potential of this tool is supported by an excellent reliability. Sensitivity to change ability of OCT is under evaluation to determine whether the technique could be used as outcome measure of skin involvement in SSc. Disclosure of Interest None Declared

SAT0467 The Five Prospective Observational Trials of the International Systemic Sclerosis FP7-Health Research Project Desscipher: A Interim Report

Background Due to lack of adequate clinical research data, drug treatment of the rare disease systemic sclerosis (SSc) is commonly off-label. The international EC-funded research project DeSScipher (acronym for “to decipher the optimal management of systemic sclerosis”) was designed to increase the evidence-based treatment strategies for SSc patients and subsequently to improve their long-term quality-of-life. Objectives The primary objective is to compare the outcomes of different treatments with respect to efficacy and safety of currently used off-label drugs from the early to the advanced phases of the main SSc-associated organ dysfunctions in a routine rheumatology in- and outpatient setting. Methods Five prospective observational trials (OTs), carried out within the EULAR Scleroderma Trials and Research (EUSTAR) group, have been designed to analyze current treatment approaches of early functionally relevant manifestations such as digital ulcers (OT1) and hand arthritis (OT2) to the morbidity and mortality-driving manifestations such as interstitial lung disease (OT3), pulmonary hypertension (OT4) and severe heart disease (OT5). The study protocols are accessible at clinicaltrials.gov Identifiers NCT01836263, NCT01834157, NCT01858259, NCT01840748, NCT01829126. Results Between April 2013 and January 2015, 1781 SSc patients have been screened at 27 contributing EUSTAR centers. 1577 (89%) patients have been enrolled into at least one of the five OTs. In particular, 1179, 127, 981, 237 and 716 patients have been enrolled into OT1-5, respectively (3240 in total; a given patient could be enrolled into multiple OTs), which represents a baseline patient recruitment rate of 79% of the target number of 4098 patients (accordingly 226%, 79%, 59%, 25% and 91% of the required number of 522, 160, 1670, 960, 786 patients for OT1-5, respectively). The completion of 1-year (OT3, OT5) and 2-year follow-up visits (OT1, OT2, OT4) are pending. Conclusions DeSScipher is currently the largest prospective observational research project ever for SSc. While patient recruitment is still ongoing, preliminary results of the five OTs are expected in late 2015 and the final results depending on the completion of follow-up visits are expected between 2017 and 2018. Acknowledgements The DeSScipher project was funded by the European Communitys Framework Programme 7 (FP7-HEALTH-2012.2.4.4-2 - Observational trials in rare diseases) under grant agreement N° 305495. We acknowledge the contribution of the following EUSTAR centers: Wuppertal (member N°192), Lille (93), Bad Bramstedt (187), Moscow (78), Assiut (168), Moscow (190), Bucharest (100), Monserrato (142), Iasi (162), Cluj-Napoca (16), Frankfurt (124), Salford/Manchester (80), Tübingen (56), Ancona (34), Zagreb (51) and Roma (94). Disclosure of Interest None declared

OP0305 Assessment of Skin Involvement by Acoustic Radiation Force Impulse (ARFI) Imaging in Patients with Systemic Sclerosis

Background Measurement of skin involvement is essential for diagnosis and assessment of prognosis in systemic sclerosis (SSc). The modified Rodnan Skin Score (mRSS) is the gold standard outcome measure for assessment of skin thickness by palpation. The mRSS has been criticised for being associated with high inter-observer variability and requiring a skilled trained investigator. There is a need therefore for a reliable and objective method to assess skin stiffness. Elastography Ultrasound (EUS) is a new, non-invasive method, which has allowed qualitative and quantitative measurements of relative skin stiffness. Recently, acoustic radiation force impulse (ARFI) with a new 3rd generation technology, virtual touch quantification (VTQ), has added a new dimension, reporting absolute values for the wave propagation speed (absolute tissue stiffness). The use of VTQ technology has never before been reported in patients with SSc. Objectives To assess skin absolute stiffness in SSc using VTQ. Methods Skin absolute stiffness was measured by VTQ at 5 of the 17 anatomical sites of the mRSS (forearm, hand, phalanx, leg and foot, of the dominant limb). Eight patients and eight age and gender matched controls were included in the study. Ultrasound measurements of absolute skin stiffness were compared with mRSS. Absolute skin stiffness measurements are presented as median plus interquartile ranges. Spearman correlation tests were used to calculate associations between ultrasound measurements and mRSS. Results Absolute skin stiffness measurements were systematically higher in SSc patients vs controls in 4 out of 5 measurements sites: forearm [2.4 (0.5) vs 1.7 (0.3)], hand [3.0 (1.4) vs 2.0 (0.9)], phalanx [3.8 (3.0) vs 2.1 (0.5)] and dorsum of the foot [4.4 (3.3) vs 3.1 (1.9)]. Forearm absolute skin stiffness correlated with the total mRSS (r=0.707, p=0.002). Hand absolute skin stiffness correlated with the local mRSS for this site of analysis (r=0.609, p=0.012).figure 1 VTQ of the volar aspect of the forearm in a control (A) and in a patient with SSc (B). Skin absolute stiffness values (shear-wave velocities) are presented (m/s) (right side). Red indicates stiff tissue, green/yellow intermediate stiffness and blue low stiffness. Tissues with a higher shear-wave velocity have higher stiffness. Image/graph Conclusions VTQ is a novel and promising application of ARFI imaging. It provides a very precise quantification of absolute skin stiffness and has potential as an objective measure for skin assessment. Further studies are warranted to define the potential contribution of VTQ to the clinical evaluation and for its use as potential outcome measure of skin involvement in SSc. References Clements PJ, et al Arthritis Rheum.1990;Iagnocco A, et al J Rheumatol. 2010;Di Geso L, et al Clin Exp Rheumatol. 2011. Disclosure of Interest None Declared

Epithelial cells undergoing epithelial mesenchymal transition (EMT) in systemic sclerosis lack caveolin-1 and modulate WNT signaling in the dermis by secreting SFRP4

Background Systemic sclerosis is a chronic fibrotic disease highly heterogeneous in clinical outcome, involving autoimmune activation, fibroproliferative vasculopathy and tissue fibrosis of skin and multiple internal organs. The mechanisms linking immune activation and tissue fibrosis are still not fully characterised. A widely accepted model of immune-mediated skin fibrosis is chronic sclerodermoid graft versus host disease (Scl-GVHD), a form of chronic GVHD in patients receiving allogenic bone marrow transplant. Histopathologic studies of cGVHD skin biopsies confirmed the presence of both fibroproliferative vasculopathy and tissue fibrosis in Scl-GVHD. Objective To identify which genes are differentially expressed in SSc skin biopsies are similarly expressed in the transcriptome of Scl-GVHD skin biopsies and therefore of potential importance in linking immune activation and skin fibrosis. Methods Metanalysis of microarray data published in the literature identified a set of 80 genes whose differential expression is highly reproduced in our SSc skin biopsies signature. The mRNA expression level of these genes was then analysed in eight Scl-GVHD and three cGVHD skin biopsies, and compared to normal skin and their differential expression in SSc. Genes found to be significantly differentially expressed (p<0.05) in univariate analysis were tested in multivariate analysis. To validate our mRNA findings we preformed immunofluorescence studies on skin biopsies. Results 46 genes were differentially expressed in cGVHD biopsies and 34 remained unique of SSc. 78.3% of the differentially expressed genes had a similar pattern of regulation in SSc. 25% were similarly expressed in both cGVHD variants, whereas 16.6% were specific of Scl-GVHD. Remarkably, this analysis identified several chemokines (CCL5, CXCL9-10-11) specifically involved in the fibrotic versus non-fibrotic response in GVHD and also increased expression of SFRP4, a potent angiogenesis inhibitor, in SSc and Scl-GVHD. Using confocal microscopy we identified as the source of increased SFRP4, cells in the basal layer of the epidermis which lost E-cadherin expression, co-expressed vimentin and specifically lacked Caveolin-1 expression. Conclusion Approximately 50% of SSc signature genes presented an altered expression in cGVHD. The further characterisation of the cells that play a role both in the fibrotic process, by undergoing EMT, and in the vasculopathy, by inhibiting angiogenesis through SFRP4 inhibition of WNT signalling, may pave the way to understand the link between these two processes in SSc.

Three-dimensional nail imaging by optical coherence tomography: a novel biomarker of response to therapy for nail disease in psoriasis and psoriatic arthritis

of local heat therapy in treating chromoblastomycosis may not only depend on its direct impact on the fungus. Enhancement of local immunity against the fungal infection, as proposed by Takahashi et al., probably plays an important role. Additionally, increasing the temperature of the local environment may cause denaturation of skin proteins such as keratin and collagen, which may impede adherence of the fungus to epithelial tissue and prevent its further growth. Local heat therapy for treating chromoblastomycosis has been reported for over 30 years, but this remedy is frequently ignored in clinical practice nowadays, possibly due to a lack of standard methodology. However, we believe heat therapy at 40 °C for 8 h/day to be an effective and tolerable therapeutic option for patients with cutaneous chromoblastomycosis. We hope this traditional remedy with few AEs may find its way back to the clinical practice again.

FRI0443 Digital artery volume index: the first objective, automated, non-invasive imaging diagnostic of macrovascular involvement in ssc

Background Macrovascular involvement in Systemic Sclerosis (SSc) is caused by proliferation of vascular smooth muscle cells within the intima of arteries (neointimal proliferation). The resulting decrease in arterial volume is responsible for the severity of Raynaud’s and for the onset of severe clinical manifestations such as Renal Crisis and Pulmonary Artery Hypertension. Several RCTs have demonstrated the efficacy of Endothelin Receptor Antagonists (ERAs) in targeting neointimal proliferation, which makes them to date, the only disease modifying agent available in SSc. Nevertheless, the lack of validated early diagnostics means the intention to treat with ERAs remains limited to the diagnosis of end stage clinical manifestations of neointimal proliferation, such as presence of Digital Ulcer (DU) and PAH. Objectives Here we aimed to determine the proof of concept validity of automated Digital Artery Volume Index (DAVIX) measured by non-contrast Time of Flight (TOF) MRI as an objective diagnostic tool to be used as surrogate outcome measure of neointimal proliferation in SSc. Methods 10 Healthy Volunteers (HV) and 8 SSc patients were enrolled. Six patients underwent longitudinal assessments at least 12 months apart. MRI scans were performed on a 3T Magnetom Verio (Siemens) and consisted of a VIBE 3D T1 scan and a 2D TOF sequence of 8 min. DAVIX was calculated as the percentage of the ratio of digital artery and the respective finger volumes. The vessels and fingers were manually outlined by an expert radiologist and used as a ‘gold standard’ (DAVIX-1) and compared to a 2nd independent radiologist assessment (DAVIX-2). An automated segmentation algorithm was developed using threshold based segmentation and region growing (DAVIX-A) and validated against gold standard. Intraclass correlation coefficient (ICC) and absolute agreement were calculated to estimate reliability. Bland-Altman bias and 95% limits of agreement (LoA) were calculated as well. Results SSc patients and HV had comparable age (44±8 vs 45±8) and gender (F:M=5:2 vs 4:2). 4 fingers were affected by DUs at baseline and two fingers were affected by new DU at follow up. Mean(±SD) DAVIX in HV was 1.21 (±0.39) with no significant difference among individual fingers. Mean DAVIX in SSc patients was 0.37 (±0.18) and 0.32 (±0.39) at baseline and follow-up, respectively (p<0.0001 vs HV for both). The fingers with DU had an average DAVIX of 0.24 (±0.08) vs 0.40 (±0.18) of the fingers without DU (p=0.02). DAVIX changed over time in both directions. In the fingers affected by new DU, DAVIX dropped by 56% (0.161 vs 0.072) and 86% (1.149 vs 0.132), respectively. ICC among two independent readings was 0.90 (95% LoA −0.139, 0.118) with overall r2=0.74 (p<0.0001). Automated segmentation showed superior correlation to gold standard with r2=0.80 (p<0.0001). Conclusions This proof of concept study demonstrated validity and sensitivity to change of DAVIX for the automated volumetric assessment of digital arteries, which reflected clinical worsening in patients with new DU. Larger, longitudinal studies are planned to assess DAVIX’ predictive value for the onset of DU and its potential use as early diagnostic of neointimal proliferation in SSc. Disclosure of Interest None declared